Generating Chimeric Zebrafish Embryos by Transplantation

Summary

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

Abstract

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

Protocol

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection ....

Discussion

The ease with which transplantation can be used to produce targeted chimeras is one of the great powers of the zebrafish as a vertebrate model. A modification of this protocol not described above allows analysis of maternal gene function for genes with essential roles in zygotic development. Since these mutant fish cannot survive until adulthood, it is necessary to transfer the mutant germline into an otherwise wild-type host embryo, creating “germline clones” of mutant cells. Generating germline mosaics in.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Injection rig	Applied Scientific Instrumentation	MPPI-3	Foot pedal can be ordered separately
Pronase	Sigma-Aldrich	P5147	protease type XIV”
Pen-Strep	Sigma-Aldrich	P4458	100x stock
Methyl cellulose	Sigma-Aldrich	M-0387
Fluorescent dextrans	Molecular Probes, Life Technologies	various	Eg. lysine-fixable rhodamine dextran (D7162)
Injection pipettes (1.2mmx0.94mmx10cm)	Sutter Instrument Co.	BF120-94-10
Transplant pipette option 1	World Precision Instruments, Inc.	TW100-4
Transplant pipette option 2	VWR international	#53508-400
micromanipulator	Narishige International	UMJ-3FC (?)
Micromanipulator magnetic stand	Narishige International	GJ-1
Transplant apparatus	Sutter Instrument Co.	MI-10010
Fine micromanipulator	Narishige International	MMO-203
Depression slides	Fisher Scientific	12560A
Agar molds for injection and transplants	AL-AN Mfg, Inc.
Reagents: Recipes for many of the reagents used for these procedures are found in The Zebrafish Book, which is available in full online at http://zfin.org/zf_info/zfbook/zfbk.html. Recipes are provided there for preparation of Fish Water, Embryo Medium with and without antibiotics, pronase for dechorionating, fluorescent dextrans, and methyl cellulose.